1. Field of the Invention
The present invention relates to a novel gluconate dehydratase capable of efficiently producing a 2-keto-3-deoxyaldonic acid from aldonic acid, to a base sequence encoding the gluconate dehydratase, to a plasmid containing the base sequence, and to a cell transformed by the plasmid. The present invention also relates to a process with use of the gluconate dehydratase or the transformed cell for producing a 2-keto-3-deoxyaldonic acid and a 2-deoxyaldonic acid or 2-deoxyaldose whose carbon number is reduced by 1.
2. Description of the Related Art
2-keto-3-deoxyaldonic acids are useful as pharmaceutical material. For example, 2-keto-3-deoxyaldonic acids are decarboxylated to 2-deoxyaldonic acids or 2-deoxyaldoses whose carbon number is reduced by 1. These substances are thought of for raw materials of antibiotics, antiviral agents, antisense drugs, and other drugs and medicines.
On the other hand, enzymes have been known which catalyze a reaction for producing 2-keto-3-deoxyaldonic acids by dehydration of aldonic acids. For example, a gluconate dehydratase (EC4. 2. 1. 39) for dehydrating D-gluconic acid to synthesize 2-keto-3-deoxy-D-gluconic acid is derived from Clostridium pasteurianum (Analytical Biochemistry 61, 275 (1974)), Alcaligenes sp. strain M250 (Methods in Enzymology 41, 99 (1975)), or Sulfolobus solfataricus (Biotechnol. Lett. 8,497 (1986)). Unfortunately, it is reported that the gluconate dehydratase derived from Clostridium pasteurianum is liable to oxidize with air and is thus rapidly deactivated in the presence of air. The gluconate dehydratase derived from Alcaligenes sp. strain M250 is not thermally stable. A report has taught that the dehydration activity of the gluconate dehydratase is 50% degraded in storage at 0° C. for 7 days in a tris(hydroxymethyl)aminomethane (hereinafter referred to as Tris) buffer solution (pH 8.0 to 8.8) containing 1 mM of sodium D-gluconate and 1 mM of magnesium chloride. Hence, these microorganisms or enzymes derived from these microorganisms are not suitable for industrial production because of the difficulty in handling, such as degradation of the reactivity in a process. The gluconate dehydratase derived from Sulfolobus solfataricus has not yet purified, and accordingly, its scientific characteristics have not been known. For synthesis of 2-keto-3-deoxy-D-gluconic acid with use of the Sulfolobus solfataricus as it is, this microorganism has activity of decomposing the product 2-keto-3-deoxy-D-gluconic acid, thus causing the yield to decrease.
As described above, no gluconate dehydratase industrially applicable in practice has been discovered, and no industrial process for efficiently producing a 2-keto-3-deoxyaldonic acid has been established.